This project investigates the most likely cause for high mutation rates within certain portions of the HIV-1 genome, inaccurate DNA synthesis by the HIV-1 reverse transcriptase (RT). Most mistakes made in vitro are initiated by strand slippage. In order to gain insight into why the HIV-1 RT is so error-prone for these types of mistakes, we are examining mutant derivatives of the RT, with emphasis on amino acids believed to be important for template-primer interactions. Mutants with alanine substituted for four specific amino acids in alpha helix H of the thumb subdomain and one palm residue all have reduced DNA binding affinity, resistance to AZTTP, reduced processivity and/or reduced framshift fidelity. These phenotypes are consistent with molecular modeling studies suggesting that these five amino acids comprise a "hydrophobic minor groove tracking module" that contacts the template-primer just back from the active site. We have also analyzed mutants in the I helix and shown that single changes in this element are kinetically silent, in contrast to the predictions of the "helix-clamp" concept in the literature. Analysis of these wild-type and mutant reverse transcriptases will enhance our understanding of how RTs interact with their substrates and may provide insights into the hypermutability of the AIDS virus. Since these studies focus on mutations resulting from strand slippage, they may also provide insights relevant to several human diseases characterized by mutations that may result from strand slippage during DNA replication.